We propose to maximize the utility of liposomes as a carrier for the cytoplasmic delivery of various macromolecules. We plan to achieve this by the introduction of new procedures which fall into three categories. 1) Cytoplasmic release of liposome contents after endocytosis: This will be enhanced by constructing pH-sensitive liposomes that fuse with the endocytic membranes when exposed to the low pH environment following endocytosis, or ii) by osmotic lysis of pinosomes after endocytosis of liposomes in hyperosmotic media. 2) Internalization of ligand-specific liposomes: We will conjugate to the liposome surface antibodies and other proteins which recognize specific cells-surface receptors. Advantage will be taken of receptors that are internalized rapidly, and also of liposomes that recognize more than one receptor. 3) Fusion with the plasma membranes induced by membrane-active compounds: We will optimize the conditions for fusion induced by PEG, tocopherol, PVA, DMSO and other fusogenic compounds, while minimizing their cytotoxic effects. The cytoplasmic release of liposome contents will be followed by especially chosen fluorescent probes (calcein and fluorescinated dextran) and encapsulated colloidal gold. These microscopic probes are normally localized either on the cell surface or in the secondary lysosomes. Their cytoplasmic localization will be used as a guide for optimization of the proposed procedures. The delivery of functionally intact macromolecules will be followed by cytotoxicity assays using encapsulated methotrexate-Gamma-aspartate as well as diphtheria toxin A fragment, and by infectivity assays using viral (SV40) DNA. These biological probes will also serve as useful models for cellular incorporation of a) non-permeant pharmacological agents, b) enzymes and proteins and c) foreign genes and other recombinant DNA.